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HOOK2 / Protein Hook homolog 2 · IHC design guide
Design Immunohistochemistry for HOOK2
Plan paraffin-section HOOK2 IHC using the reported cytoplasmic tissue pattern (HPA tissue IHC). Compare strong staining in glandular cells with the absence of detectable staining in adipocytes, and assess localization alongside intensity (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HOOK2 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic staining in tissue sections (HPA tissue IHC) | |
| Staining pattern | Widespread cytoplasmic staining across cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A08854-1) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adipose tissue |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A08854-1) | |
| Caveat | Adipocytes may show no detectable staining (HPA tissue IHC) | |
| Regulation | Low tissue specificity; no regulator reported (HPA tissue IHC) | |
| Isoform / epitope | 2 isoforms; confirm epitope coverage (UniProt) |
Section 1
Recommended HOOK2 IHC & IF Protocols
The catalog antibody uses EDTA pH 8.0 retrieval (datasheet A08854-1). The published HOOK2 IHC methods below describe free-floating brain sections (PMC4370497) and antibody dilutions for endogenous and overexpressed Hook proteins (PMC1896156).
| Sample | Paraffin-embedded human ovarian cancer tissue; fixative not specified (datasheet A08854-1) |
| Fixation | Image fixative and duration unreported (datasheet A08854-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A08854-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A08854-1) |
| Primary antibody | Rabbit anti-HOOK2, 2-5μg/ml (datasheet A08854-1) |
| Primary incubation | Overnight at 4 °C (datasheet A08854-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A08854-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | HOOK2-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A08854-1); citrate pH 6 retrieval was reported for free-floating brain sections (PMC4370497).
Section 2
What Is the Expected HOOK2 Staining Pattern?
HOOK2 should appear mainly in the cytoplasm of many cell types, including strongly stained glandular cells and kidney tubule cells (HPA tissue IHC: ubiquitous cytoplasmic expression; High in listed cells). Perinuclear or punctate signal is plausible (UniProt Q96ED9: centrosome, trans-Golgi network and cytoplasmic foci). HOOK2 has no transmembrane segment, so a crisp plasma-membrane outline is unexpected (UniProt Q96ED9 topology). HPA rates the tissue IHC profile Approved, with medium consistency against RNA data (HPA tissue IHC).
| Chromogenic cytoplasmic staining is High in colon glandular cells or kidney tubule cells; nearby cell-free areas remain clear. | This matches two HPA positive examples (HPA tissue IHC: High in colon glandular cells and kidney tubule cells). Compare cells within the section rather than requiring every cell to stain equally: the profile is ubiquitous, while the reported intensity varies by cell type (HPA tissue IHC: profile and staining levels). |
| Fine perinuclear or punctate cytoplasmic staining accompanies broader cytoplasmic signal; ciliary rootlets stain in fallopian tube or nasopharynx. | These locations can fit HOOK2 biology and observed tissue staining (UniProt Q96ED9: centrosome, trans-Golgi network and cytoplasmic foci; HPA tissue IHC: High in ciliated-cell rootlets). A punctum alone is less convincing than a pattern with the expected cells and a clean control (general IHC interpretation practice). |
| Signal is chiefly nuclear or forms a sharp plasma-membrane rim, with little cytoplasmic staining. | Treat this as a discordant compartment pattern and investigate artefact or nonspecific binding (HPA tissue IHC: ubiquitous cytoplasmic expression; UniProt Q96ED9: cytoplasmic locations and no transmembrane segment). Chromogenic morphology alone cannot identify which staining step caused the discrepancy (general IHC interpretation practice). |
| Adipocytes stain strongly while expected positive cells do not, or colour appears in a section lacking primary antibody. | Strong adipocyte staining conflicts with the reported HPA example, where adipocytes are Not detected (HPA tissue IHC: adipose tissue). Colour without primary antibody points to endogenous detection activity or reagent background; a tissue pattern alone cannot distinguish cross-reactivity from detection artefact (general IHC control interpretation). |
| Uniform haze covers tissue and cell-free spaces, or an expected positive section has no detectable signal. | Haze extending beyond cells is background rather than interpretable HOOK2 localisation (general IHC interpretation practice). No signal in colon glandular cells or kidney tubules conflicts with HPA positive examples, but does not by itself prove HOOK2 absence; assay performance needs checking first (HPA tissue IHC: High in both). |
💡Expected HOOK2 appearanceCall a result convincing when staining is predominantly cytoplasmic and High in an HPA positive cell population such as colon glandular cells or kidney tubules (HPA tissue IHC: profile and High examples); discrete perinuclear signal can fit (UniProt Q96ED9), whereas dominant nuclear staining or colour across cell-free spaces warrants investigation (general IHC interpretation practice).
| Tissue and cell selection | Use a named positive cell population for interpretation: appendix, colon, duodenum, gallbladder and rectum glandular cells, kidney tubules, and ciliated-cell rootlets in fallopian tube or nasopharynx are High (HPA tissue IHC). Adipocytes are a useful contrasting population because HPA reports them as Not detected (HPA tissue IHC). |
| Compartment and topology | HOOK2 is associated with cytoplasm, centrosome, microtubules and trans-Golgi network; cytoplasmic foci are also reported (UniProt Q96ED9). It has no transmembrane segment (UniProt Q96ED9 topology). Judge a focal pattern in its cell context; the protein record does not make every isolated punctum specific. |
| IHC antibody evidence | HPA043519 and HPA050351 each have IHC status Approved (HPA antibodies). The supplied record does not label either antibody IHC Enhanced (HPA antibodies). HPA describes medium consistency between tissue staining and RNA data, so use the listed cell patterns as reference observations, not an absolute rule for every section (HPA tissue IHC). |
| IF/ICC Q: What localisation is reported? | HPA reports vesicles and cytosol as supported ICC-IF locations (HPA subcellular). UniProt also reports centrosome, trans-Golgi network and cytoplasmic foci (UniProt Q96ED9). Those sources describe localisation; they do not provide an IF/ICC protocol for this IHC section. |
| Isoforms and modifications | UniProt lists 2 isoforms and phosphorylation sites at residues 163, 230 and 710 (UniProt Q96ED9). The supplied sources do not establish which isoforms or modified states the IHC antibodies detect. Avoid assigning intensity differences to isoforms or phosphorylation without separate validation. |
| Situation | Likely cause | Next action |
|---|---|---|
| No stain in colon glandular cells or kidney tubules. | The result misses HPA High reference populations (HPA tissue IHC); a failed staining run remains possible (general IHC practice). | Confirm the positive cells are present, then check primary-antibody use, chromogen development and run controls before interpreting the section as HOOK2 negative (general IHC practice). |
| Strong stain in adipocytes, especially when expected positive cells are weak. | HPA reports adipocytes as Not detected (HPA tissue IHC); nonspecific binding or detection background may explain discordant colour (general IHC practice). | Compare a no-primary control and an HPA positive cell population in the same run; reassess antibody dilution and blocking if the discrepancy persists (general IHC practice). |
| Dominant nuclear staining or a continuous membrane rim. | The pattern conflicts with cytoplasmic HOOK2 localisation and absent transmembrane topology (HPA tissue IHC; UniProt Q96ED9 topology). | Check the counterstain and no-primary control; repeat with the IHC-validated antibody and compare cellular compartments in a known positive tissue (general IHC practice; HPA antibodies: IHC Approved). |
| Brown haze extends through tissue and cell-free regions. | A spatially uniform deposit is compatible with background from blocking, washing or detection chemistry (general IHC practice). | Review the no-primary control; improve washing or blocking and inspect chromogen development before scoring cytoplasmic signal (general IHC practice). |
| Only tiny perinuclear dots appear in an otherwise negative section. | Centrosomal or punctate localisation is plausible (UniProt Q96ED9), but dots alone do not establish antibody specificity (general IHC interpretation practice). | Check whether dots occur in HPA positive cell populations, then compare with the no-primary control and broader cytoplasmic staining (HPA tissue IHC: positive cells; general IHC practice). |
| Ciliary rootlet signal is absent in fallopian tube or nasopharynx. | HPA reports High staining in ciliated-cell rootlets at both sites (HPA tissue IHC), so missing signal may reflect sampling or assay performance (general IHC practice). | Verify that ciliated cells and their rootlets are represented in the section; assess the run against another HPA positive population before calling the tissue negative (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing HOOK2 staining
A08854-1 · IHCIHC analysis of HOOK2 using anti-HOOK2 antibody (A08854-1). HOOK2 was detected in a paraffin-embedded section of human ovarian cancer tissue
A08854-1 · IHCIHC analysis of HOOK2 using anti-HOOK2 antibody (A08854-1). HOOK2 was detected in a paraffin-embedded section of human placenta tissue. Heat
A08854-1 · IFIF analysis of HOOK2 using anti-HOOK2 antibody (A08854-1). HOOK2 was detected in immunocytochemical section of A431 cells. Enzyme antigen re
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000095066-HOOK2/tissue/cerebral+cortex#imid_14060922
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000095066-HOOK2/tissue/colon#imid_14060848
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000095066-HOOK2/tissue/liver#imid_14060857
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000095066-HOOK2/tissue/kidney#imid_14060842
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA050351; green = HOOK2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA050351; green = HOOK2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HOOK2 IHC & IF
🧪Run colon first and expect staining in its glandular cells (HPA: High in colon glandular cells). Run adipose tissue as the negative, focusing on adipocytes (HPA: Not detected in adipocytes); on the colon slide, use non-glandular cells as an internal background comparator without assuming they are HOOK2-negative.
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show HOOK2 in A-431, MCF-7, U2OS, NIH 3T3, with annotated localisation: Vesicles (supported), Cytosol (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and concentration-matched nonimmune rabbit IgG isotype controls, plus HOOK2-knockout tissue or peptide competition when a validated blocking peptide is available (standard IHC practice; caption: rabbit primary antibody). For chromogenic colon IHC, quench endogenous peroxidase and check for signal in the no-primary control (standard IHC practice; caption: HRP/DAB detection).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A08854-1 tissue-IHC caption does not state the fixative (caption: fixative not stated). Heat retrieval in EDTA at pH 8.0 was used for the shown paraffin section; that example does not establish whether retrieval is required (caption: EDTA retrieval). IF/ICC is documented in cell-line images with supported vesicular and cytosolic localization, but the supplied evidence does not show that frozen sections or IF are easier than paraffin IHC; in colon, assess glandular-cell staining separately from luminal deposits (HPA: ICC-IF images and supported locations; HPA: High in colon glandular cells; standard IHC interpretation).
HPA tissue IHC evidence for HOOK2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Fallopian tube | Ciliated cells (ciliary rootlets) | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced HOOK2 IHC Tips
Troubleshoot HOOK2 staining in paraffin sections by checking retrieval, cytoplasmic localisation, cell type, and chromogenic controls before scoring.
How should I retrieve HOOK2 antigen in paraffin sections?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 (datasheet A08854-1). This is the retrieval condition documented for the catalog antibody in a paraffin-embedded human ovarian cancer section (datasheet A08854-1). Keep section thickness, heating, and cooling consistent across comparison slides, then assess staining alongside a no-primary control (standard IHC practice). If signal is weak, first check that the EDTA solution reached the intended pH and that sections remained covered during heating (standard IHC practice). Change one retrieval variable at a time and compare cytoplasmic signal with tissue background (standard IHC practice; HPA: ubiquitous cytoplasmic expression).
Could fixation explain weak HOOK2 staining?
Target-specific sensitivity to fixation is unknown from the supplied evidence (datasheet A08854-1: fixative not stated). The catalog image documents a paraffin-embedded section, but its caption does not identify a fixative or fixation duration (datasheet A08854-1). Record those details for each specimen and compare sections processed under matched conditions before attributing a staining difference to HOOK2 abundance (standard IHC practice). Run the documented EDTA pH 8.0 retrieval and 2 μg/ml primary concentration consistently while assessing the discrepancy (datasheet A08854-1; standard IHC practice). Include a well-preserved comparison section to help distinguish poor morphology from weak staining (standard IHC practice).
Which staining pattern should count as HOOK2 localisation?
Expect predominantly cytoplasmic staining, potentially including perinuclear or punctate signal, when assessing chromogenic sections (HPA: ubiquitous cytoplasmic expression; UniProt Q96ED9: centrosome, trans-Golgi network, and cytoplasmic foci). Supported cell-imaging locations include vesicles and cytosol (HPA: subcellular localisation). Strong nuclear-only staining deserves scrutiny because the supplied localisation records do not identify the nucleus as a HOOK2 location (UniProt Q96ED9; HPA: subcellular localisation). Compare the pattern within intact cells against the no-primary slide and surrounding tissue background (standard IHC practice). In ciliated epithelium, assess ciliary rootlets separately from diffuse cytoplasm, since high rootlet staining is reported in fallopian tube and nasopharynx (HPA: tissue IHC).
Can this stain distinguish HOOK2 isoforms or phosphorylation states?
HOOK2 has 2 annotated isoforms, but the supplied catalog caption does not map the antibody epitope (UniProt Q96ED9; datasheet A08854-1). The protein includes a calponin-homology domain at residues 6–122 and annotated phosphorylation at residues 163, 230, and 710 (UniProt Q96ED9). Those annotations alone cannot establish which isoform or modification state produces chromogenic staining (UniProt Q96ED9; datasheet A08854-1). Check immunogen and isoform sequence coverage before making an isoform-specific claim, and use an independently validated modification-specific reagent if phosphorylation is the question (standard IHC practice). Score this assay as total detectable HOOK2 unless epitope-level evidence supports a narrower interpretation (datasheet A08854-1).
How should I follow up an IHC pattern with HOOK2 immunofluorescence?
For a separate IF/ICC experiment, pair HOOK2 with a marker for the expected cell type, such as a ciliated-cell marker when examining reported rootlet staining (HPA: fallopian tube and nasopharynx). Select spectrally separated fluorophores and place the weaker signal in a far-red channel if tissue autofluorescence obscures shorter wavelengths (standard IF practice). HOOK2 lacks a transmembrane segment and has cytoplasmic locations, so permeabilise cells or sections to access intracellular epitopes while checking that the chosen treatment preserves puncta (UniProt Q96ED9; standard IF practice). Compare the resulting cytosolic and vesicular pattern with supported cell-imaging locations, using single-label and no-primary controls for bleed-through and background (HPA: subcellular localisation; standard IF practice).
How can I reduce diffuse brown background without losing HOOK2 signal?
First compare the stained section with a no-primary control to separate primary-dependent signal from background in the chromogenic workflow (standard IHC practice). The catalog example used 10% goat serum blocking, 2 μg/ml rabbit primary overnight at 4°C, and a peroxidase-conjugated secondary for 30 minutes at 37°C (datasheet A08854-1). Check endogenous peroxidase blocking, wash stringency, secondary specificity, and DAB development time if brown staining persists without primary antibody (standard IHC practice). Titrate primary concentration around the documented condition only after retrieval and controls are consistent (datasheet A08854-1; standard IHC practice). Judge improvements by preserved cellular cytoplasmic staining relative to background (HPA: tissue IHC; standard IHC practice).
How should I quantify HOOK2 staining across tissue samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, and exclude folds, necrosis, and poorly preserved edges (standard IHC practice). For cellular staining, report the percentage of positive cells and an H-score from 0–300, calculated from the percentages at intensity grades 0–3 (standard IHC practice). For punctate staining, report positive-punctum density per mm² of evaluable tissue or per counted cell, with the denominator stated (standard image-analysis practice). Normalise comparisons to the same cell type, evaluable area, staining run, and acquisition settings (standard IHC practice). Broad cytoplasmic expression and only medium RNA–protein consistency make cell-type-specific scoring especially useful here (HPA: tissue IHC, Approved).
What separates convincing HOOK2 staining from an artefact?
A convincing result places signal within intact cellular cytoplasm and, where resolved, near the centrosome or in puncta (HPA: ubiquitous cytoplasmic expression; UniProt Q96ED9: centrosome and cytoplasmic foci). High staining in glandular cells or ciliary rootlets is compatible with reported tissue patterns, while adipocytes were not detected in the cited tissue profile (HPA: tissue IHC). Treat staining concentrated at section edges, necrotic zones, or damaged tissue as suspect until it reproduces in intact regions (standard IHC practice). Brown signal on a no-primary section warrants checking endogenous peroxidase and detection reagents before assigning it to HOOK2 (standard IHC practice). Confirm any unexpected nuclear-only or extracellular pattern with an independent specificity control (UniProt Q96ED9; standard IHC practice).
Boster reagents
Best HOOK2 / Protein Hook homolog 2 IHC Antibodies
A08854-1 has IHC images from human ovarian cancer and placenta paraffin sections and an IF/ICC image from A431 cells (catalog image captions).
Real IHC data
Anti-HOOK2 Antibody ®
Cat # A08854-1
A08854-1 is the SKU with a rendered card: its IHC figure shows a human ovarian cancer paraffin section (A08854-1 IHC caption). Its catalog also documents IHC in human placenta and IF/ICC in A431 cells (A08854-1 image captions).
Which to pick: For tissue IHC, choose A08854-1: its rabbit antibody has a documented paraffin-section result using EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody; the fixative is unreported (A08854-1 IHC caption). For IF/ICC, choose A08854-1 because its A431 image documents staining at 5 μg/ml; M08854 lists IHC but has no supplied IHC figure or IF/ICC application (A08854-1 IF caption; M08854 catalog entry). Neither SKU has documented cross-species reactivity; both list Human only (catalog reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q96ED9 (HOOK2_HUMAN, Protein Hook homolog 2).
- Human Protein Atlas. HOOK2 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. HOOK2 subcellular location (ICC-IF): Localized to vesicles and cytosol..
- Human Protein Atlas. HOOK2 antibody validation summary (2 antibodies).
- Hook proteins: association with Alzheimer pathology and regulatory role of hook3 in amyloid beta generation. PloS one 2015 — PMC4370497.
- Hook2 contributes to aggresome formation. BMC cell biology 2007 — PMC1896156.
- PubMed PMID:9927460 — UniProt-cited evidence.
- PubMed PMID:15057824 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HOOK2 (Protein Hook homolog 2). Expected localisation Cytoplasmic staining in tissue sections (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A08854-1). Validated IHC antibody: A08854-1 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human ovarian cancer tissue; fixative not specified (datasheet A08854-1); 2. Fixation: Image fixative and duration unreported (datasheet A08854-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: EDTA pH 8.0 (datasheet A08854-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A08854-1); 8. Primary antibody: Rabbit anti-HOOK2, 2-5μg/ml (datasheet A08854-1); 9. Primary incubation: Overnight at 4 °C (datasheet A08854-1). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A08854-1); Caveat: Adipocytes may show no detectable staining (HPA tissue IHC); Isoform / epitope: 2 isoforms; confirm epitope coverage (UniProt); Regulation: Low tissue specificity; no regulator reported (HPA tissue IHC). Evidence: UniProt Q96ED9, HPA tissue IHC · Approved, HPA ICC-IF · vesicles and cytosol., Datasheet A08854-1, PMC4370497, PMC1896156. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HOOK2/immunohistochemistry-ihc-guide.